Abstract Title: Molecular Mechanisms Underlying Oseteopontin Isoform-Induced Neovascularization Coronary artery and peripheral arterial disease (CAD and PAD) remain leading causes of mortality in the United States and both result in ischemia downstream of vessel occlusion sites. The body's endogenous response to ischemia is to increase neovascularization to recover blood flow through the formation of a functional collateral vessel network. One of the mechanisms by which collateral vessel formation occurs is through arteriogenesis, which is defined as the enlargement of existing capillaries and arterioles into larger conductance arteries downstream of vessel occlusion sites. Arteriogenesis requires cytokine signaling, macrophage recruitment, matrix remodeling, and Vascular Smooth Muscle Cell (VSMC) proliferation and migration. We previously demonstrated that osteopontin (OPN), a secreted, multifunctional, glycol-phospho-protein that can act as a cytokine, is highly upregulated in response to ischemia and is a critical mediator of collateral vessel formation. OPN primarily signals through CD44 and integrin receptors and has been linked to cell survival, proliferation, migration and adhesion, all of which are required for collateral formation. Unlike rodents, humans expresses three OPN isoforms as a result of alternative splicing: OPNa, OPNb, and OPNc. Our preliminary data demonstrate that these human OPN (hOPN) isoforms have divergent effects on functional collateral formation in vivo and cell migration in vitro, despite intact integrin and CD44 receptor binding domains in all 3 isoforms. However, how OPN isoforms differentially promote collateral vessel formation and cell migration and what different OPN isoforms are expressed in human ischemic tissues remain undefined. OPN is subject to extensive post-translational modifications (PTMs), including phosphorylation and glycosylation, which could have significant implications for its intrinsic biological function. Therefore, we hypothesize that differences in phosphorylation of OPN isoforms may significantly alter OPN isoform receptor binding to CD44 and/or ?v integrins and, thus, contribute to differences in OPN isoform intrinsic biological function in cell migration and neovascularization. This project will determine determine the OPN phosphorylations necessary for OPN isoform-specific receptor binding to promote OPN-induced VSMC migration and collateral formation. Understanding the mechanisms by which human OPN isoforms differentially promote collateral formation and cell migration could ultimately provide mechanic insights into the development of novel therapeutic targets to treat patients with obstructive arterial disease, enhancing blood flow and retain tissue function.